Piezo Channels Research Articles

Efficient coarse-grained simulations of multiple Piezo channels in flat membrane and vesicles.

The unique dome shape of mechanosensitive Piezo channels has inspired biophysical studies on whether and how Piezos should cluster under various cell types and physiological conditions. Molecular dynamics (MD) simulation can, in principle, capture Piezo diffusion and clustering stemming from interactions at the near atomistic level. However, due to their very large size, sampling spontaneous inter-channel Piezo interactions is inaccessible by standard MD techniques based on all-atom or Martini coarse-grained (CG) force fields. To overcome this challenge, we developed a minimal CG model for simulating the dynamics of multiple Piezo channels at the submicron scale in membranes and vesicles. In addition, our model enables tuning the shape and mechanical properties of both channel and membrane. Using this model, we investigated the propensity and topology of Piezo clustering under different channel densities, protein conformations, membrane bending rigidity, and membrane curvature. This work provides new insights into the interplay between protein and membrane deformation energy on Piezo clustering, hence bridging the gap between continuum elasticity theory and high-resolution imaging techniques.

The energetics of rapid cellular mechanotransduction.

Cells throughout the human body detect mechanical forces. While it is known that the rapid (millisecond) detection of mechanical forces is mediated by force-gated ion channels, a detailed quantitative understanding of cells as sensors of mechanical energy is still lacking. Here, we combine atomic force microscopy with patch-clamp electrophysiology to determine the physical limits of cells expressing the force-gated ion channels Piezo1, Piezo2, TREK1, and TRAAK. We find that, depending on the ion channel expressed, cells can function either as proportional or nonlinear transducers of mechanical energy, detect mechanical energies as little as ∼100 fJ, and with a resolution of up to ∼1 fJ. These specific energetic values depend on cell size, channel density, and cytoskeletal architecture. We also make the surprising discovery that cells can transduce forces either nearly instantaneously (<1ms), or with substantial time delay (∼10 ms). Using a chimeric experimental approach and simulations we show how such delays can emerge from channel-intrinsic properties and the slow diffusion of tension in the membrane. Overall, our experiments reveal the capabilities and limits of cellular mechanosensing and provide insights into molecular mechanisms that different cell types may employ to specialize for their distinct physiological roles.

Eicosapentaenoic acid enhances PIEZO2 inactivation and counteracts PIEZO2 gain of function mutations.

PIEZO2 is an essential mechanosensitive ion channel for touch discrimination, vibration, interoception, and proprioception. Mechanosensitive ion channels rely on membrane composition to transduce physical stimuli into electrical signals. Dietary fatty acids are among the membrane lipid components that dynamically regulate ion channel function. We have previously shown that when part of the plasma membrane, the polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (C20:5, EPA) decreases membrane rigidity and bending stiffness, while enhancing PIEZO1 inactivation. To determine if EPA could also modulate PIEZO2 channel function, we used electrophysiology, fatty acid supplementation, dietary intervention, and lipidomics in vitro and ex vivo. We found that EPA, when incorporated into the membrane, increased the inactivation of heterologously expressed PIEZO2 without significantly affecting the current magnitude or apparent activation threshold. Similarly, we measured an increase in PIEZO2 inactivation in rat and mouse dorsal root ganglia (DRG) neurons after EPA supplementation. Importantly, cultured DRG neurons from mice carrying the C. elegans FAT-1 enzyme (which converts ω-6 to ω-3 PUFAs) or fed for 4-12 weeks with an EPA-enriched diet displayed enhanced PIEZO2 inactivation when compared to neurons from control mice. Moreover, using a consecutive mechanical step protocol, we found that PIEZO2 slowly recovered from inactivation in DRG neurons supplemented with EPA, from fat-1 mice, or from mice fed with an EPA-enriched diet, when compared with the control. These results support the notion that EPA favors PIEZO2 non-conductive states. Gain-of-function PIEZO2 mutations causing Distal Arthrogryposis type 5 display decreased PIEZO2 inactivation and enhanced recovery from inactivation. We show that EPA supplementation restored the gating properties of Distal Arthrogryposis causing mutations to wild-type levels in vitro. Overall, our findings show that dietary fatty acids modulate PIEZO2 function and could be an effective strategy to ameliorate GOF PIEZO2-mediated diseases.

Piezo2 mediates visceral mechanosensation: A new therapeutic target for gut pain?

Mechanical distension/stretch in the colon provokes visceral hypersensitivity and pain. In this issue of Neuron, Xie etal. report that mechanosensitive Piezo2 channels, expressed by TRPV1-lineage nociceptors, are involved in visceral mechanical nociception and hypersensitivity.

Mechanosensitive ion channel PIEZO1 and rare genetic disorders.

Lower urinary tract symptoms (incontinence, urgency, frequency) cost the US $10 billion annually. Detrusor underactivity (DU) is a weak bladder contraction with slow, incomplete bladder emptying that often requires bladder emptying by catheter ± surgeries since drug therapies are ineffective. DU affects all ages by myogenic or neurogenic etiologies, leading to UTIs, stones, hydronephrosis ± vesicoureteral reflux, renal insufficiency/failure. Barriers exist to progress in treatment, with poor genetics and mechanistic understanding, and few animal models of disease.

PIEZO1 activates non-canonical EGFR internalization and signaling.

EGFR-ERK signaling controls cell cycle progression during development, homeostasis, and disease. Both the soluble extracellular ligand, EGF, and mechanical inputs like matrix stiffness, cell adhesion, or stretch can activate EGFR-ERK signaling. However, the molecules transducing mechanical forces into EGFR signaling remain unidentified. We previously found that stretch promotes mitosis by mechanically activating the ion channel Piezo1 to trigger ERK signaling. Here, we show that Piezo1 provides the missing link to mechanical EGFR-ERK activation. Both EGF or the synthetic Piezo1 agonist Yoda1 triggered EGFR internalization and ERK activation, established markers of EGFR signaling. However, while EGF stimulation requires canonical EGFR tyrosine autophosphorylation, activation by Piezo1 requires non-canonical serine EGFR phosphorylation by a Src-p38 kinase axis. Additionally, Yoda1 stimulation of Piezo1 promoted cell cycle re-entry and proliferation via long-term nuclear accumulation of ERK, AP-1 (FOS and JUN), and YAP, typical of regenerative and malignant signaling. Our results suggest two modes of EGFR signaling: basal EGF-dependent signaling, requiring tyrosine autophosphorylation, and mechanically activated EGFR signaling, that results in sustained proliferation, critical to repair and regeneration, and cancer growth.

A novel activator of Piezo1: Electrophysiological study.

Mechanosensitive ion channel Piezo1 plays important roles in diverse physiological processes, including differentiation, angiogenesis, vasodilation, wound healing, and inflammation. To date, three Piezo1 activators (Yoda1, Jedi1, and Jedi2) were discovered through high-throughput screening. These are artificially synthesized chemicals and not pharmacologically authorized, thus having potentials to cause problems when being administrated in human. In this study, we introduce a novel activator of Piezo1 (NAP1) that is a natural component of organisms. The effect of NAP1 on Piezo1 current was tested through electrophysiological recording on Piezo1-deficient (P1KO) HEK293T cells with or without transfection of human Piezo1 (hPiezo1). Without hPiezo1 on P1KO HEK293T, mechanical stimulation failed to activate current, which was still not observable after the application of NAP1 to the cells. However, NAP1 sensitized hPieoz1 currents in various patch configurations. In cell-attached recording, extracellular NAP1 (10, 30, and 100 µM) increased hPiezo1 currents in a dose-dependent manner, shifted the current-stimulus curve leftwards, and increased the pressure sensitivity. However, 100 µM NAP1 didn’t alter the single channel conductance of hPiezo1. In outside-out and whole-cell recordings, NAP1 reversibly enhanced hPiezo1 currents. The association and dissociation times of 100 µM NAP1 measured in the outside-out configuration were approximately 8 seconds. Like Yoda1, NAP1 did not sensitize human Piezo2 in the whole cell configuration. Since NAP1 is biocompatible and orally administrable, it will replace the other Piezo1 activators, with its biocompatibility triggering the vitalization of animal studies involving Piezo1.

PIEZO1 regulates leader cell formation and cellular coordination during collective cell migration: An integrative multiscale modeling and experimental study.

The collective migration of keratinocytes during wound healing requires both the generation and transmission of mechanical forces for individual cellular locomotion as well as for the coordination of movement across cells. Leader cells initiated along the wound edge transmit mechanical and biochemical cues to ensuing follower cells, ensuring their uniform polarization and coordinated direction of migration, or directionality. Despite the observed importance of mechanical cues in leader cell formation and governing directionality, the underlying biophysical mechanisms remain elusive. The mechanically activated ion channel PIEZO1 was recently identified to play an inhibitory role during the reepithelialization of wounds through retraction of keratinocytes located along the wound edge. Here, through an integrative experimental and mathematical modeling approach, we elucidate PIEZO1's contributions to collective migration. Time-lapse microscopy reveals that PIEZO1 activity inhibits leader cell formation along the wound edge. To probe the relationship between PIEZO1 activity, leader cell formation and inhibition of reepithelialization, we developed an integrative 2D-multiscale model of wound closure that links observations at the single cell and collective cell migration scales. Through numerical simulations and subsequent experimental validation, we found that directionality plays a key role during wound closure and is inhibited by PIEZO1 activity. We propose that PIEZO1-mediated retraction suppresses leader cell formation which inhibits the coordination of directionality between cells during collective migration.

Single-particle tracking and machine-learning classification reveals heterogeneous Piezo1 diffusion.

The mechanically-activated ion channel Piezo1 is involved in numerous physiological processes. Piezo1 is activated by diverse mechanical cues and is gated by membrane tension. The channel has been found to be mobile in the plasma membrane. We employed single particle tracking (SPT) of endogenously-expressed, tdTomato-tagged Piezo1 using Total Internal Reflection Fluorescence Microscopy in two cell types, mouse embryonic fibroblasts and liver sinusoidal endothelial cells. Application of SPT unveiled a surprising heterogeneity of Piezo1 mobility in the plasma membrane. Leveraging a machine learning technique, Piezo1 trajectories were sorted into distinct classes (“mobile,” “intermediate,” and “trapped”) by partitioning features that describe the geometric properties of a trajectory. To evaluate the effects of the plasma membrane properties on Piezo1 diffusion, we manipulated membrane composition by depleting or supplementing cholesterol or by adding margaric acid to stiffen the membrane. To address effects of channel activation on Piezo1 mobility, we treated cells with Yoda1, a Piezo1 agonist, and GsMTx-4, a channel inhibitor. We collected thousands of trajectories for each condition, and found that “mobile” Piezo1 in cells supplemented with cholesterol or margaric acid exhibited decreased mobility, whereas Piezo1 in cholesterol-depleted membranes demonstrated increased mobility, compared to their respective controls. Additionally, activation by Yoda1 increased Piezo1 mobility and inhibition by GsMTx-4 decreased Piezo1 mobility compared to their respective controls. The “mobile” trajectories were analyzed further by fitting the time-averaged mean-squared displacement as a function of lag time to a power-law model, revealing Piezo1 consistently exhibits anomalous subdiffusion. This suggests Piezo1 is not freely mobile, but that its mobility may be hindered by subcellular interactions. These studies illuminate the fundamental properties governing Piezo1 diffusion in the plasma membrane and set the stage to determine how specific cellular interactions may influence channel activity and mobility.

A computation model of the multi-layer brain-tumor-blood barrier structure under mechanical load.

Mechanical forces are abundant within the tumor microenvironment, including the solid stress generated from tumor growth and shear stress from the blood flow and the cerebellum spinal fluid. In brain tumors, the blood-tumor barrier (BTB), which consists of brain tumor-initiating cells (BTIC), pericytes, basement membrane, and endothelial cells, limits the access of most therapeutic agents to the tumor. Our previous work showed brain tumor-initiating cells are sensitive to mechanical signals, and knocking down the mechanical sensitive ion channel Piezo2 disrupts the BTB in medulloblastoma. Understanding how mechanical forces are transmitted to the BTB is important to decipher the mechanism by which BTB integrity is regulated. In this work, a multi-layer computation model, which is composed of BTICs, pericytes, basement membrane, and endothelial cells, was constructed based on transmission electron microscopy images of the BTB. The mechanical properties of each layer were set as isotropically elastic with Young's modulus of 0.5 kPa for BTIC, 0.5 kPa for pericyte, 20 kPa for basement membrane, and 3 kPa for endothelial cell. The force generated by pericytes was applied to pericyte's edge, mimicking the force generated at the edge during contraction of the pericyte. The force generated by the pericyte was assigned as 7 nN, calculated based on traction force microscopy measurement. To study the stress distribution under mechanical loading, a spherical indenter with a radius of 1.5 µm was added to the top of the BTB structure to apply a force of 10 pN. The results showed that the pericyte-generated stress is transmitted to all cellular and extracellular BTB components and the basement membrane converts pericyte contraction into compressive stress applied onto the endothelial cell. When subjected to mechanical stimulation, the basement membrane bears most mechanical stress within the BTB.

Gut touch: Molecules, cells, circuits, and purpose.

Most animals conduct the entire digestive process within the GI tract while keeping the luminal contents entirely outside the body, separated by the tightly sealed GI epithelium. Therefore, like the skin and oral cavity, the GI tract must sense the chemical and physical properties of luminal contents at this interface to perfect function. Specialized sensory enteroendocrine cells (EECs) in the GI epithelium interact intimately with luminal contents. Recently, we discovered an EEC subpopulation that expresses mechanically gated ion channel Piezo2 and is developmentally and functionally like the population of skin's touch sensors - the Merkel cells. Given these similarities, we hypothesized that the gut has intrinsic tactile sensitivity, which we call gut touch. The Piezo2+ EEC mechanotransduction mechanism involves voltage-gated calcium and sodium ion channels and intracellular calcium stores to amplify and prolong the cytoplasmic calcium elevation, which drives the release of signaling molecules such as serotonin. The released EEC signaling molecules activate multiple downstream intrinsic sensory endings that drive intrinsic reflex motility and secretion circuits. I will end by exploring the physiologic purpose of gut touch and how gut touch disruptions lead to human disease.

Improved PIEZO1 agonism through 4-benzoic acid modification of Yoda1.

The protein PIEZO1 forms mechanically activated, calcium-permeable, non-selective cation channels in numerous cell types from several species. Options for pharmacological modulation are limited and so we modified a small-molecule agonist at PIEZO1 channels (Yoda1) to increase the ability to modulate these channels. Medicinal chemistry generated Yoda1 analogues that were tested in intracellular calcium and patch-clamp assays on cultured cells exogenously expressing human or mouse PIEZO1 or mouse PIEZO2. Physicochemical assays and wire myography assays on veins from mice with genetic disruption of PIEZO1. A Yoda1 analogue (KC159) containing 4-benzoic acid instead of the pyrazine of Yoda1 and its potassium salt (KC289) have equivalent or improved reliability, efficacy and potency, compared with Yoda1 in functional assays. Tested against overexpressed mouse PIEZO1 in calcium assays, the order of potency (as EC50 values, nM) was KC289, 150 > KC159 280 > Yoda1, 600). These compounds were selective for PIEZO1 over other membrane proteins, and the physicochemical properties were more suited to physiological conditions than those of Yoda1. The vasorelaxant effects were consistent with PIEZO1 agonism. In contrast, substitution with 2-benzoic acid failed to generate a modulator. 4-Benzoic acid modification of Yoda1 improves PIEZO1 agonist activity at PIEZO1 channels. We suggest naming this new modulator Yoda2. It should be a useful tool compound in physiological assays and facilitate efforts to identify a binding site. Such compounds may have therapeutic potential, for example, in diseases linked genetically to PIEZO1 such as lymphatic dysplasia.

The role of PIEZO ion channels in the musculoskeletal system.

PIEZO1 and PIEZO2 are mechanosensitive cation channels that are highly expressed in numerous tissues throughout the body and exhibit diverse, cell-specific functions in multiple organ systems. Within the musculoskeletal system, PIEZO1 functions to maintain muscle and bone mass, sense tendon stretch, and regulate senescence and apoptosis in response to mechanical stimuli within cartilage and the intervertebral disc. PIEZO2 is essential for transducing pain and touch sensations as well as proprioception in the nervous system, which can affect musculoskeletal health. PIEZO1 and PIEZO2 have been shown to act both independently as well as synergistically in different cell types. Conditions that alter PIEZO channel mechanosensitivity, such as inflammation or genetic mutations, can have drastic effects on these functions. For this reason, therapeutic approaches for PIEZO-related disease focus on altering PIEZO1 and/or PIEZO2 activity in a controlled manner, either through inhibition with small molecules, or through dietary control and supplementation to maintain a healthy cell membrane composition. Although many opportunities to better understand PIEZO1 and PIEZO2 remain, the studies summarized in this review highlight how crucial PIEZO channels are to musculoskeletal health and point to promising possible avenues for their modulation as a therapeutic target.

Microscopic mechanism of PIEZO1 activation by pressure-induced membrane stretch.

Mechanosensitive PIEZO1 ion channels open in response to membrane stretch. Yet, the underlying microscopic mechanism of this activation remains unknown. To probe this mechanism, we used cell-attached pressure-clamp recordings to measure single channel currents at different steady-state negative pipette pressures, spanning the full range of the channel's pressure sensitivity. Pressure-dependent activation occurs through a sharp reduction of the mean shut duration and through a moderate increase of the mean open duration. Across all tested pressures, the distribution of open and shut dwell times best follows sums of two and three exponential components, respectively. As the magnitude of the pressure stimulus increases, the time constants of most of these exponential components gradually change, in opposite directions for open and shut dwell times, and to a similar extent. In addition, while the relative amplitudes of fast and slow components remain unchanged for open intervals, they fully reverse for shut intervals, further reducing the mean shut duration. Using two-dimensional dwell time analysis, Markov-chain modeling, and simulations, we identified a minimal five-states model which recapitulates essential characteristics of single channel data, including microscopic reversibility, correlations between adjacent open and shut intervals, and asymmetric modulation of dwell times by pressure. This study identifies a microscopic mechanism for the activation of PIEZO1 channels by pressure-induced membrane stretch and deepens our fundamental understanding of mechanotransduction by a vertebrate member of the PIEZO channel family.

Molecular taxonomy of nociceptors and pruriceptors.

Molecular taxonomy of nociceptors and pruriceptors.

The mechanosensitive ion channel Piezo1 modulates the migration and immune response of microglia

Microglia are the brain's resident immune cells, performing surveillance to promote homeostasis and healthy functioning. While microglial chemical signaling is well-studied, mechanical cues regulating their function are less well-understood. Here, we investigate the role of the mechanosensitive ion channel Piezo1 in microglia migration, pro-inflammatory cytokine production, and stiffness sensing. In Piezo1 knockout transgenic mice, we demonstrated the functional expression of Piezo1 in microglia and identified genes whose expression was consequently affected. Functional assays revealed that Piezo1 deficiency in microglia enhanced migration toward amyloid β-protein, and decreased levels of pro-inflammatory cytokines produced upon stimulation by lipopolysaccharide, both invitro and invivo. The phenomenon could be mimicked or reversed chemically using a Piezo1-specific agonist or antagonist. Finally, we also showed that Piezo1 mediated the effect of substrate stiffness-induced migration and cytokine expression. Altogether, we show that Piezo1 is an important molecular mediator for microglia, its activation modulating microglial migration and immune responses.

FM1-43 Dye Memorizes Piezo1 Activation in the Trigeminal Nociceptive System Implicated in Migraine Pain.

It has been proposed that mechanosensitive Piezo1 channels trigger migraine pain in trigeminal nociceptive neurons, but the mechanosensitivity of satellite glial cells (SGCs) supporting neuronal sensitization has not been tested before. Moreover, tools to monitor previous Piezo1 activation are not available. Therefore, by using live calcium imaging with Fluo-4 AM and labeling with FM1-43 dye, we explored a new strategy to identify Piezo channels' activity in mouse trigeminal neurons, SGCs, and isolated meninges. The specific Piezo1 agonist Yoda1 induced calcium transients in both neurons and SGCs, suggesting the functional expression of Piezo1 channels in both types of cells. In Piezo1-transfected HEK cells, FM1-43 produced only a transient fluorescent response, whereas co-application with Yoda1 provided higher transient signals and a remarkable long-lasting FM1-43 'tail response'. A similar Piezo1-related FM1-43 trapping was observed in neurons and SGCs. The non-specific Piezo channel blocker, Gadolinium, inhibited the transient peak, confirming the involvement of Piezo1 receptors. Finally, FM1-43 labeling demonstrated previous activity in meningeal tissues 3.5 h after Yoda1 washout. Our data indicated that trigeminal neurons and SGCs express functional Piezo channels, and their activation provides sustained labeling with FM1-43. This long-lasting labelling can be used to monitor the ongoing and previous activation of Piezo1 channels in the trigeminal nociceptive system, which is implicated in migraine pain.

Mechanosensitive channel Piezo1 is an essential regulator in cell cycle progression of optic nerve head astrocytes.

Optic nerve head (ONH) astrocytes provide structural and metabolic support to neuronal axons in developmental, physiological, and pathological progression. Mechanosensitive properties of astrocytes allow them to sense and respond to mechanical cues from the local environment. We confirmed that ONH astrocytes express the mechanosensitive ion channel Piezo1 in vivo. By manipulating Piezo1 knockdown or overexpression in vitro, we found that Piezo1 is necessary but insufficient for ONH astrocyte proliferation. Loss of Piezo1 can lead to cell cycle arrest at G0/G1 phase, a possible mechanism involving decreased yes-associated protein (YAP) nuclear localization and downregulation of YAP-target cell cycle-associated factors, including cyclin D1 and c-Myc. Gene ontology enrichment analysis of differential expression genes from RNA-seq data indicates that the absence of Piezo1 affects biological processes involving cell division. Our results demonstrate that Piezo1 is an essential regulator in cell cycle progression in ONH astrocytes.

Transcranial Magneto-Acoustic Stimulation Attenuates Synaptic Plasticity Impairment through the Activation of Piezo1 in Alzheimer's Disease Mouse Model.

The neuropathological features of Alzheimer's disease include amyloid plaques. Rapidly emerging evidence suggests that Piezo1, a mechanosensitive cation channel, plays a critical role in transforming ultrasound-related mechanical stimuli through its trimeric propeller-like structure, but the importance of Piezo1-mediated mechanotransduction in brain functions is less appreciated. However, apart from mechanical stimulation, Piezo1 channels are strongly modulated by voltage. We assume that Piezo1 may play a role in converting mechanical and electrical signals, which could induce the phagocytosis and degradation of Aβ, and the combined effect of mechanical and electrical stimulation is superior to single mechanical stimulation. Hence, we design a transcranial magneto-acoustic stimulation (TMAS) system, based on transcranial ultrasound stimulation (TUS) within a magnetic field that combines a magneto-acoustic coupling effect electric field and the mechanical force of ultrasound, and applied it to test the above hypothesis in 5xFAD mice. Behavioral tests, invivo electrophysiological recordings, Golgi-Cox staining, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, real-time quantitative PCR, Western blotting, RNA sequencing, and cerebral blood flow monitoring were used to assess whether TMAS can alleviate the symptoms of AD mouse model by activating Piezo1. TMAS treatment enhanced autophagy to promote the phagocytosis and degradation of β-amyloid through the activation of microglial Piezo1 and alleviated neuroinflammation, synaptic plasticity impairment, and neural oscillation abnormalities in 5xFAD mice, showing a stronger effect than ultrasound. However, inhibition of Piezo1 with an antagonist, GsMTx-4, prevented these beneficial effects of TMAS. This research indicates that Piezo1 can transform TMAS-related mechanical and electrical stimuli into biochemical signals and identifies that the favorable effects of TMAS on synaptic plasticity in 5xFAD mice are mediated by Piezo1.

Electro-mechanical coupling directs endothelial activities through intracellular calcium ion deployment.

Conversion between mechanical and electrical cues is usually considered unidirectional in cells with cardiomyocytes being an exception. Here, we discover a material-induced external electric field (Eex) triggers an electro-mechanical coupling feedback loop in cells other than cardiomyocytes, human umbilical vein endothelial cells (HUVECs), by opening their mechanosensitive Piezo1 channels. When HUVECs are cultured on patterned piezoelectric materials, the materials generate Eex (confined at the cellular scale) to polarize intracellular calcium ions ([Ca2+]i), forming a built-in electric field (Ein) opposing Eex. Furthermore, the [Ca2+]i polarization stimulates HUVECs to shrink their cytoskeletons, activating Piezo1 channels to induce influx of extracellular Ca2+ that gradually increases Ein to balance Eex. Such an electro-mechanical coupling feedback loop directs pre-angiogenic activities such as alignment, elongation, and migration of HUVECs. Activated calcium dynamics during the coupling further modulate the downstream angiogenesis-inducing eNOS/NO pathway. These findings lay a foundation for developing new ways of electrical stimulation-based disease treatment.